Plant Cell, Tissue and Organ Culture 20: 131-135, 1990 © 1990Kluwer Academic Publishers. Printed in the Netherlands Short communication
Influence of cultural and physiochemical factors on ascorbate stability in plant tissue culture media H. Wayne Elmore, Barbara Samples, Sanjeev Sharma & Marcia Harrison Department of Biological Sciences, Marshall University, Huntington, W V 25701, USA
Received30 May 1988;acceptedin revisedform 17 October 1989
Key words: L-ascorbic aid; dehydroascorbic acid; tissue culture; medium Abstract
Ascorbic acid rapidly decays in plant tissue culture media. Within 50 min to 3 h after preparing 100 mM solutions, ascorbic acid was destroyed. Autoclaving, shaking flasks, high light intensity and increasing pH over a range from 4.5-7 accelerated decay. Ascorbic acid was oxidized to dehydroascorbic acid which also underwent decay. Within 11 h and 15 min after adding ascorbic acid both ascorbic acid and its oxidation product, dehydroascorbic acid, disappeared from medium. Since ascorbic acid is rapidly destroyed in plant tissue culture media it may not exert its effect as an intact molecule. Instead its antioxidant/antibrowning role in plant cell, tissue and organ cultures may be mediated by some product of further oxidation.
Introduction
Ascoribc acid has been frequently included in culture medium to improve growth of algae [1], plant cell suspensions [2] and tissue cultures [3, 4]. In vitro ascorbic acid has been shown to act as an antioxidant/antibrowning agent [2-4]. Browning in plant tissues has been attributed to polyphenol oxidase [5]. This enzyme has a broad substrate specifity and acts through a two-step reaction catalysing the o-hydroxylation of monophenols and the subsequent oxidation of o-diphenols to o-diquinones [6]. This latter reaction which results in the formation of strongly-oxidizing quinones thought to cause poor growth and browning [7] is inhibited by ascorbate [8]. The modes of action attributed to ascorbic acid would require the presence of reduced ascorbate [8]. However, evidence exists suggesting that ascorbic acid is unstable in beverages, food samples and aqueous solutions [9, 10]. Ascorbic acid is oxidized by reactions catalysed by Cu(II) and Fe(III) [11], both of which are components of plant tissue culture media. Temperature [9], dissolved oxygen [10] and pH [9] also affect the stability of ascorbic aid.
This investigation studied the stability of ascorbic acid within the milieu of plant tissue culture medium and the effects of cultural and environmental parameters on its rate of decay.
Materials and methods Cultural conditions. All experiments were conducted in 250 ml flasks containing 100 ml Murashige & Skoog's [13], Brown's [14], Knudson's [15] medium or distilled water. Water was glass-distilled and deionized using a Millipore Model 2932 organic carbon unit and two Model 2931 mixed-bed deionizing units in series, pH was adjusted to 5.8 immediately after addition of L-ascorbic acid, medium was not autoclaved and flasks were held at 25 °C in a water bath under ambient fluorescent light of approximately 1.61 W m 2 unless otherwise stated. Effects of light on ascorbate destruction was tested using 150 W photoflood lamps. Ascorbic acid assay. Experiments were initiated by
adding sufficient ascorbic acid to yield 100 mM.
132 This concentration was chosen since it was within the range used in plant tissue culture media [2] and because concentrations up to 100 mM yield a linear standard curve [17]. Ascorbic acid was assayed using a Varian DHA 90UV-visible spectrophotometer at 265 nm according to Meucci et al. [9]. Three replicate samples were analysed for each assay and experiments were repeated. The standard error of the mean was calculated but not included since in most cases the error bars did not extend beyond the symbols used to indicate points.
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Dehydroascorbic acid assay. When ascorbic acid is oxidized, dehydroascorbic acid is the product [18]. Dehydroascorbic acid formed from the oxidation of ascorbic acid added to medium was assayed after its reduction to ascorbic acid using a method modified from Schreiber et al. [19]. Experiments were initiated by adding ascorbic acid. At intervals, absorbance at 265 nm was measured. Sufficient dithiothreitol to yield 10 mM was then added to reduce dehydroascorbic acid to ascorbic acid. After 15 min, absorbance at 265 nm was measured again. The difference between absorbance before and after the addition of dithiothreitol was taken as a measure of dehydroascorbic acid present. Thus, the rates of ascorbic acid decay, dehydroascorbic acid formation and dehydroascorbic acid decay were followed.
Results and discussion
The decay of ascorbic acid was followed in three common plant tissue culture media and glassdistilled, deionized water. Within 50 min after adding ascorbic acid it had decayed in Murashige & Skoog's and Brown's media (Fig. 1). The rate of decay was slower in distilled water but the slowest rate of decay was observed in Knudson's medium which contained detectable amounts of ascorbic acid for almost 3 h. The differences in the rates of decay in culture media might be attributed to different concentrations of Cu(II) and Fe(III), known to catalyse the oxidation of ascorbic acid [11]. Differences in water purified and deionized by different methods were shown to affect the stability of ascorbic acid differently depending on purity [12]. However no satisfactory explanation can account for the apparent greater stability of ascorbic acid in
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Knudson's medium which contains Cu(II) and Fe(III). From these media, Murashige & Skoog's was selected for subsequent experiments on the detailed study of cultural and environmental parameters on the rate of decay since it is extensively used and commercially available (Gibco, Madison, Wis., USA). The results of experiments that tested the effects of a pH range (4.5-7) demonstrated that increasing pH accelerated decay. Ascorbic acid was most stable at pH 4.5 (Fig. 2). At pH 7, ascorbic acid was destroyed within 20 min. Most plant tissue culture media are routinely adjusted between pH 5.5 and 6.5. Within this range, ascorbic acid was detectable for 50-75 min after it was added. Medium containing ascorbic acid has been routinely autoclaved [2, 16], however, studies have reported the temperature-dependent destruction of ascorbic acid [12]. To test the effects of autoclaving on ascorbate destruction flasks containing 100 nM
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Flasks were shaken at 150rpm and the control remained stationary during the experiment. Shaking flasks slightly accelerated the rate of ascorbic acid decay, resulting in its destruction within 70 min, approximately 15 min sooner than in control flasks (data not shown). Effects of light on the rate of ascorbic acid destruction were studied. Light intensity ranging from 0-8.05Wm -2 did not affect the rate of ascorbic acid decay, but 16.1Wm -2 slightly accelerated destruction resulting in the disappearance of ascorbic acid within 46min, approximately 5 min before the dark control (data not shown). Since ascorbic acid disappeared rapidly from plant tissue culture medium, a study of its conversion to the oxidation product, dehydroascorbic acid, was undertaken. When ascorbic acid at 100 mM was added to Murashige & Skoog's medium it began its rapid decay. As the concentration of ascorbic acid decreased, the levels of dehydroascorbic acid concurrently increased reaching a maximum at a time that coincided with the point at which ascorbic acid disappeared (Fig. 3). Dehydroascorbic acid then began a decline that continued over approximately 11 h. By 11 h and 15 min after adding 100 mM ascorbic acid, no detectable ascorbic acid nor dehydroascorbic acid remained.
Fig. 2. Effect ofpH on the rate of ascorbic acid decay in Murashige & Skoog's medium, pH 4.5 (o--o); pH 5 (O---O); pH 5.5 (A--A); pH 6 (zx--,x); pH 6.5 (~ ~); pH 7 (D---U).
ascorbic acid were prepared, initial absorbance measured and the flasks divided into two groups. The first group was placed in darkness at 25 °C. The second group was autoclaved at l kgcm -2 for 15 min. Cooling of medium to 25 °C required 5 min. Approximately 49% and 12% of the ascorbic acid remained in the unautoclaved and autoclaved flasks, respectively, at the end of the 20 min experiment (data not shown). Autoclaving dramatically accelerated the rate of destruction. The level of oxygen in stored foods affects the level of ascorbic acid [10]. Frequently, flasks containing cultured plant cells are placed on a shaker to increase the levels of dissolved oxygen. Since the destruction of ascorbic acid is the result of its oxidation [11], the effects of shaking were studied.
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Fig. 3. Decay of ascorbic acid (o--o), appearance and the decay of dehydroascorbic acid ( ~ ) in Murashige & Skoog's medium.
134
Browning of plant tissue cultures is a serious problem in pure and applied research since it is often linked to growth inhibition and is a poorly understood phenomenon [2-4, 7, 17]. Ascorbic acid has been added as a medium supplement to decrease the deleterious effects of browning associated with poor growth [2-4, 20] and in some cases for unspecified reasons [21]. Apparently, researchers studying plant cell, tissue and organ cultures have been unaware of the rapid oxidation that ascorbic acid undergoes. Frequently, medium has been prepared, autoclaved, set aside in the light and used several hours to days later. Since ascorbic acid is an ephemeral component of plant tissue culture media it is quite possible that none exists when medium is inoculated. If medium were filter-sterilized, stored under refrigeration at low pH in air-tight containers from which oxygen had been purged, in darkness, ascorbic acid might be preserved to some degree. However, if it were brought to room temperature, the pH adjusted, inoculated and placed in light on a shaker it would undergo rapid decay to dehydroascorbic acid. Many tissues are capable of the enzymatic reduction of dehydroascorbic acid to ascorbic acid [22, 23], thus traces of ascorbic acid might be maintained. However, in actual practice it is more likely that both ascorbic acid and dehydroascorbic acid would be destroyed by the time medium could be used. The rapid oxidation of ascorbic acid suggests that it may not exert its effects directly as an intact molecule nor is it likely that dehydroascorbic acid remains in medium sufficiently long to allow its uptake by tissues and reduction to ascorbic acid. Alternatively, the effects may be mediated by some product of further oxidation.
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